Theurapeutic or prophyiactic agent for arthritis

ABSTRACT

This invention provides a new therapeutic or prophylactic agent for arthritis such as osteoarthritis. Specifically, it provides a therapeutic or prophylactic agent for arthritis such as osteoarthritis, or an agent for promoting the growth of articular chondrocyte, comprising a guanyl cyclase B (GC-B) activator as an active ingredient; or a method for inhibiting arthritis or for promoting the growth of articular chondrocyte by activating GC-B; or a method for screening an agent for promoting the growth of articular chondrocyte or an agent capable of treating arthritis using the GC-B activity as an indication.

TECHNICAL FIELD

The present invention relates to a therapeutic or prophylactic agent forarthritis, particularly osteoarthritis and similar types of arthriticdiseases, or an agent for promoting the growth of articularchondrocytes, or a method for inhibiting the arthritis or a method forpromoting the growth of articular chondrocyte, using a guanyl cyclase B(hereinafter referred to as “GC-B”) activator. The present inventionfurther relates to a method for screening of a therapeutic agent forarthritis or an articular chondrocyte growth promoter using GC-Bactivity as an indication.

BACKGROUND ART

Arthritis is an inflammatory disease of the joint, and rheumatoidarthritis and osteoarthritis (or osteoarthrosis) are prevalent arthriticdisorders.

Rheumatoid arthritis is thought to be an autoimmune disease, accompaniedby articular pain, stiffening and swelling, and as the diseaseprogresses, may often lead to the degeneration of the articularcartilage surfaces similar to osteoarthritis, resulting in severedestruction of the articular bone and cartilage.

Osteoarthritis is a degenerative disease of the articular cartilageoccurring frequently in the elderly. Osteoarthritis (OA) involvesdestruction of the cartilage and proliferative change in the bone andcartilage resulting from degeneration of articular components, with thechange resulting in a secondary arthritis (e.g., synovitis).Osteoarthritis occurs mainly in weight-bearing joints, such as theknees, elbows and hip joints (Virchows Arch 1996; 260:521-663), and lessfrequently in non-weight-bearing joints, such as the shoulder/elbow andhand joints. Furthermore, temporomandibular arthrosis with similarconditions has been identified in the temporomandibular joint (J OrofacPain 1999; 13(4): 295-306).

It is known that the matrix proteins, which are the functional entity ofthe cartilage, are reduced, and the number of chondrocytes decreases inosteoarthritis (Arth Rheum 2002; 46(8): 1986-1996). However, due to thelack of blood vessels distributed in the cartilage tissue, the smallnumber of chondrocytes that are highly differentiated, the small numberof cartilage precursor cells, and the slow turnover of the cartilagematrix, the cartilage has too low self-reproduction ability to ensurespontaneous recovery from the decreases in articular cartilage matrixand chondrocytes in osteoarthritis (Novartis Found. Symp. 2003;249:2-16). In addition, in osteoarthritis, arthritis occurs concurrentlywith the degeneration of cartilage, leading to joint pain (J Rheumatol2001; 28(6): 1330-1337).

Examples of reported therapeutic/prophylactic agents for arthritis, suchas rheumatoid arthritis and osteoarthritis, include, for example, aprotein tyrosine kinase inhibitor (Japanese Patent Publication (Kohyo)No. 11-512708A (1999)), N-acyl-2-glucosamine derivatives (JapanesePatent Publication (Kohyo) No. 2004-507490A), and quinoline/quinazolinederivatives (Japanese Patent Publication (Kokai) No. 9-169646A (1997)).In addition, current standard therapeutic agents for osteoarthritis thathave been used widely are oral anti-inflammatory analgesics orhyaluronic acid and adrenocortical steroid preparations forintra-articular injection, which all relieve joint pain, and this meansthat drugs having inhibitory effect on the degeneration of the articularcartilage are required (Decision Base 7, 2002).

Guanyl cyclase (GC) is a membrane protein belonging to the enzyme familythat catalyzes the synthesis of the second messenger cGMP from GTP, andexamples include GC-A, GC-B, . . . , and GC-F. GC-B is found mainly invascular endothelial cells, and thought to be involved in relaxation ofthe smooth muscle. A natriuretic peptide (NP) is known to activate GC.NPs are divided into ANP (atrial sodium peptide), BNP (brain natriureticpeptide) and CNP (C-type natriuretic peptide), and they are thought toexhibit biological activity by elevating intracellular cGMP levelthrough two guanyl cyclase conjugated receptors (NPR-A for ANP and BNP,and NPR-B for CNP) (Ann Rev Biochem 1991; 60: 229-255).

NPR-C is not a guanyl cyclase conjugated receptor and thought to be aclearance receptor for NPs not involved in signal transduction (Science,1987; 238:675-678). However, in a system by which prostaglandin E₂(PGE₂) production is induced by cyclooxygenase 2 (COX-2) when mouse bonemarrow macrophages are stimulated with lipopolysaccharide (LPS), ANP andCNP have been reported to exhibit an inhibitory effect on PGE₂production by decreasing intracellular cAMP levels via NPR-C, and thissuggests the involvement of NPR-C in the signal transduction of NPs(Endocrinology 2002; 143(3): 846-852). The report describes that ANPexhibits an inhibitory effect of up to about 70% on the enhancement ofPGE₂ production through stimulation of mouse bone marrow macrophages(BMM) with LPS, while CNP exhibits only an inhibitory effect of up toabout 20%, thus CNP has a weaker effect. Because the control of COX-2production through cyclic nucleotides, such as cAMP and cGMP, is knownto represent either promotional or inhibitory reaction depending on thecell type and stimulation type, it is unclear whether the inhibition ofLPS-induced PGE₂ production in BMM cells by CNP may be applied to othercells and stimulations. In addition, Endocrinology 2002; 143(3): 846-852reported that ANP was shown to exhibit an inhibitory effect in a systemwhere LPS administration increased blood thromboxane B₂ (TXB₂) level inmice, and contrarily cANF of the same mechanism enhanced. In addition,although the report describes the application of ANP to immunity-relateddiseases, such as arthritis and sepsis, it makes no reference to theapplication of CNP to those related diseases. Consequently, no findinghas been obtained regarding the action of CNP on arthritis.

NPs have been reported to play an important role in the control ofhumoral homeostasis and blood pressure (J Clin Invest 1987;93:1911-1921, J Clin Invest 1994; 87: 1402-1412), and their expressionand biological activity in various tissues other than the cardiovascularsystem are known (Endocrinol 1991; 129:1104-1106, Ann Rev Biochem 1991;60: 553-575). For cartilage, the use of CNP for the extension ofauxotonic gristle and treatment of achondrogenesis in transgenic miceoverexpressing BNP (Proc. Natl. Acad. Sci. U.S.A. 1998; 95:2337-2342) orCNP has been reported (Nat Med 2004; 10(1): 80-86, Japanese PatentPublication (Kokai) No. 2003-113116A). However, the growth platecartilage is temporary cartilage that disappears eventually followingcalcification and displacement by bones, and it is known to havebiological properties that are different from permanent cartilage whichexists during lifetime, such as articular cartilage and trachealcartilage (Dev Biol 1989; 136(2): 500-507, J Cell Biol 2001; 153(1):87-100). Furthermore, although the in vitro activity of CNP to enhancethe hypertrophy of articular chondrocytes, which is permanent cartilage,has been reported (J Biol Chem 2003; 278(21): 18824-18832), no findinghas been obtained regarding the in vivo action on the articularcartilage in normal animals, or on the degeneration of the articularcartilage or arthritis in osteoarthritis.

In osteoarthritis, the articular cartilage swells at the earliest stageof the disease, resulting in a temporary increase in the cartilagetissue volume (J Rheum 1991; 18(3): 1905-1915), and with the progress ofthe disease, degeneration/destruction of the cartilage matrix increases,leading to a decrease in the volume (Arthritis Rheum 2004; 50(2):476-487). The number of articular chondrocytes decreases due toapoptosis (Arthritis Rheum 2004; 50(2): 507-515). On the other hand, theremaining individual articular chondrocytes are known to express type Xcollagen, and differentiate into hypertrophic chondrocytes having thenature of temporary cartilage (Arthritis Rheum 1992; 35(7): 806-811). Inaddition, arthritis accompanies the destruction of the articularcartilage and may be a factor in clinical pain in the affected joint (JRheumatol. 2001; 28(6): 1330-1337). Inhibition of these changes inosteoarthritis, i.e. the decreases in or recovery of the articularcartilage matrix and the number of articular chondrocytes, and theinhibition of arthritis, is thought to be useful in the development oftherapeutic agents.

It is an object of the present invention to provide a new therapeutic orprophylactic agent for arthritis, including osteoarthritis, or a methodfor treating the arthritis.

It is another object of the present invention to provide an agent ormethod for promoting the growth of articular chondrocytes.

It is another object of the present invention to provide a method forinhibiting arthritis including osteoarthritis.

It is another object of the present invention to provide a method forscreening of a therapeutic agent for arthritis.

It is another object of the present invention to provide a method forscreening of an agent for promoting the growth of articular chondrocyte.

SUMMARY OF THE INVENTION

We prepared a CNP transgenic mouse that overexpresses C-type natriureticpeptide (CNP), which is a kind of guanyl cyclase B (GC-B) activator, tostudy the effect on the articular cartilage, and the following resultswere obtained: in the CNP transgenic mouse, the thickness of thearticular cartilage and the number of articular chondrocytes increasedsignificantly; in an osteoarthritic model prepared from the CNPtransgenic mouse, it was resistant to articular swelling, with thedegeneration of the articular cartilage reduced, there were slightchanges in synovial cell growth, granulation and inflammatory cellinfiltration, and the proteoglycan content in articular cartilage didnot decrease, while in an osteoarthritic model prepared from a normalmouse, there were marked changes in synovial cell growth, granulationand inflammatory cell infiltration. From these findings, we have nowfound that the GC-B activator possesses anti-arthritis effect as well asassimilating action on the articular cartilage.

Therefore, the present invention comprises the following inventions.

In a first aspect, the present invention provides a therapeutic orprophylactic agent for arthritis comprising a guanyl cyclase B (GC-B)activator as an active ingredient.

In one embodiment of the present invention, the arthritis isosteoarthritis.

In another embodiment of the present invention, the osteoarthritis isosteoarthritis of weight-bearing or non-weight-bearing joints.

In another embodiment of the present invention, the osteoarthritis isdegenerative gonarthrosis.

In another embodiment of the present invention, the osteoarthritis isdegenerative coxarthrosis.

In another embodiment of the present invention, the osteoarthritis istemporomandibular arthrosis.

In another embodiment of the present invention, the arthritis is causedby rheumatoid arthritis.

In another embodiment of the present invention, the arthritis is causedby osteoarthritis.

In another embodiment of the present invention, the GC-B activator is atype C natriuretic peptide (CNP) or a derivative thereof.

In another embodiment of the present invention, the CNP described aboveis selected from CNP-22 and CNP-53 from mammals, including human, orbirds.

In another embodiment of the present invention, the CNP is CNP-22 of SEQID NO:1 or CNP-53 of SEQ ID NO:2.

In another embodiment of the present invention, the CNP derivative has adeletion, substitution or addition of one or several amino acids in theamino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, while possessing aCNP activity.

In another embodiment of the present invention, the therapeutic orprophylactic agent for arthritis further comprises at least onenonsteroidal anti-inflammatory drug.

In a second aspect, the present invention provides an agent forpromoting the growth of articular chondrocyte, comprising a GC-Bactivator as an active ingredient.

In one embodiment of the present invention, the GC-B activator is a CNPor a derivative thereof.

In another embodiment of the present invention, the CNP is CNP-22 orCNP-53 from mammals, including human, or birds.

In another embodiment of the present invention, the CNP is CNP-22 of SEQID NO:1 or CNP-53 of SEQ ID NO:2.

In another embodiment of the present invention, the CNP derivative has adeletion, substitution or addition of one or several amino acids in theamino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, while possessing aCNP activity.

In another embodiment of the present invention, the agent for promotingthe growth of articular chondrocyte further comprises at least onenonsteroidal anti-inflammatory drug.

In a third aspect, the present invention provides a method forinhibiting arthritis, wherein the arthritis is inhibited by activatingGC-B.

In one embodiment of the present invention, the GC-B is activated by aCNP or a derivative thereof.

In another embodiment of the present invention, the CNP is CNP-22 orCNP-53 derived from mammals, including human, or birds.

In another embodiment of the present invention, the CNP is CNP-22 of SEQID NO:1 or CNP-53 of SEQ ID NO:2.

In another embodiment of the present invention, the CNP derivative has adeletion, substitution or addition of one or several amino acids in theamino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, while possessing aCNP activity.

In another embodiment of the present invention, the GC-B is activated bya combination of a CNP or a derivative thereof and at least onenonsteroidal anti-inflammatory drug.

In a fourth aspect, the present invention provides a method forpromoting the growth of articular chondrocyte, wherein the growth ispromoted by activating GC-B.

In one embodiment of the present invention, the GC-B is activated by aCNP or a derivative thereof.

In another embodiment of the present invention, the CNP is CNP-22 orCNP-53 derived from mammals, including human, or birds.

In another embodiment of the present invention, the CNP described aboveis CNP-22 of SEQ ID NO:1 or CNP-53 of SEQ ID NO:2.

In another embodiment of the present invention, the derivative describedabove has deletion, substitution or addition of one or several aminoacids in the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, and hasCNP activity.

In another embodiment of the present invention, the GC-B is activated bya combination of a CNP or a derivative thereof and at least onenonsteroidal anti-inflammatory drug.

In a fifth aspect, the present invention provides a method for screeningof an agent for promoting the growth of articular chondrocyte,comprising screening candidate agents for the ability to promote thegrowth of articular chondrocyte using GC-B activity as an indication.

In one embodiment of the present invention, the method comprisespreparing cultured cells that express GC-B or cells from articularchondrocytes, culturing the cells in the presence of a candidate agent,and screening of the candidate agents for the ability to promote thegrowth of articular chondrocyte using the cellular GC-B activity as anindication.

In another embodiment of the present invention, the GC-B activity isdetermined as an amount of intracellular cGMP produced.

In another embodiment of the present invention, the method comprisespreparing a cultured cell line which has been forced to express GC-B,culturing the cell line in the presence or absence of a candidate agent,determining the amount of produced intracellular cGMP, and screening thecandidate agents for the ability to promote the growth of articularchondrocyte using as an indication the difference between the amounts ofintracellular cGMP produced in the presence and absence of the candidateagent.

In a sixth aspect, the present invention provides a method for screeninga therapeutic agent for osteoarthritis, rheumatoid arthritis orarthritis comprising screening a candidate agent for osteoarthritis,rheumatoid arthritis or arthritis using GC-B activity as an indication.

In one embodiment of the present invention, the method comprisespreparing cultured cells that express GC-B, or cells from articularchondrocytes, culturing the cells in the presence of a candidate agent,and screening the candidate agent for an agent capable of treatingosteoarthritis, rheumatoid arthritis or other arthritis using thecellular GC-B activity as an indication.

In another embodiment of the present invention, the GC-B activity isdetermined as an amount of intracellular cGMP produced.

In another embodiment of the present invention, the method comprisespreparing a cultured cell line which has been forced to express GC-B,culturing the cell line in the presence or absence of a candidate agent,determining the amount of intracellular cGMP produced, and screening ofthe candidate agent for an agent capable of treating osteoarthritis,rheumatoid arthritis or other arthritis using as an indication thedifference between the amounts of intracellular cGMP produced in thepresence and absence of the candidate agent.

In a seventh aspect, the present invention provides a therapeutic orprophylactic agent for osteoarthritis comprising a GC-B activator as anactive ingredient.

In one embodiment of the present invention, the therapeutic orprophylactic agent for osteoarthritis described above further comprisesat least one nonsteroidal anti-inflammatory drug.

In an eighth aspect, the present invention provides a therapeutic orprophylactic agent for rheumatoid arthritis comprising a GC-B activatoras an active ingredient.

In one embodiment of the present invention, the therapeutic orprophylactic agent for rheumatoid arthritis further comprises at leastone nonsteroidal anti-inflammatory drug.

In a ninth aspect, the present invention provides an activation promoterfor a guanyl cyclase B (GC-B) activator, comprising a nonsteroidalactivator.

In one embodiment of the present invention, the GC-B activator is a CNPor a derivative thereof.

In another embodiment of the present invention, the CNP is selected fromCNP-22 and CNP-53 derived from mammals, including human, or birds.

In another embodiment of the present invention, the CNP is CNP-22 of SEQID NO:1 or CNP-53 of SEQ ID NO:2.

In another embodiment of the present invention, the CNP derivative has adeletion, substitution or addition of one or several amino acids in theamino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, while possessing aCNP activity.

In another embodiment of the present invention, the nonsteroidalactivator is a cyclooxygenase inhibitor.

In another embodiment of the present invention, the cyclooxygenaseinhibitor is selected from the group consisting of indomethacin,ibuprofen, piroxicam, salicylic acid, diclofenac, ketoprofen, naproxenand piroxicam.

In a tenth aspect, the present invention further provides a method foractivating a GC-B activator, wherein the activation promoter asdescribed above is used.

The specification of this application encompasses the contents asdisclosed in the specification and/or drawings of Japanese PatentApplication No. 2004-107924, which is claimed as a priority of theapplication.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the construction of a vector for preparing a CNP transgenicmouse. FIG. 1A: cDNA of the mouse CNP, which has been incorporated intopGEM-T Easy vector, was cut out with Pst I and blunt-ended at each end.FIG. 1B: pSG1 was treated with EcoR I and blunt-ended. FIG. 1C: Themouse CNP cDNA prepared in FIG. 1A was incorporated into the pSG1obtained in FIG. 1B.

FIG. 2 shows a DNA fragment for injection. A fragment (about 2.3 kb)containing the CNP gene was cut out from pSG1-CNP prepared in FIG. 1C bydigesting with Hind III and Xho I, and it was used as a fragment forinjection.

FIG. 3 shows the results of a genotypical analysis of a CNP transgenicmouse. In the wild type mouse (WT) 3 signals (indicated as “Wild typeCNP gene”) were detected, while in the transgenic mouse (Tgm) 2 signals(indicated as “Transgene”) derived from the transgene were detected inaddition to the wild-type CNP gene.

FIG. 4 is a graph showing thickening of the articular cartilage in a CNPtransgenic mouse. The thickness of the articular cartilage of faciespatellaris femoris was compared between a normal litter (Wild) and CNPtransgenic mice (CNP tgm). The figure indicates that CNP transgenic micehave statistically significantly thicker articular cartilage. **:p<0.01, unpaired Student's t-test.

FIG. 5 is a graph showing an increase in the number of articularchondrocytes in a CNP transgenic mouse. The number of chondrocytes perfield under the optical microscope in the articular cartilage of faciespatellaris femoris was compared between a normal litter (Wild) and CNPtransgenic mice (CNP tgm). The figure indicates that CNP transgenic micehave a statistically significantly larger number of chondrocytes permicroscopic field. *: p<0.05, unpaired Student's t-test.

FIG. 6 is a graph showing resistance to articular swelling in acollagenase induced OA model in CNP transgenic mice. After administering3% collagenase or physiological saline to a CNP transgenic mouse (Tgm)and a wild-type mouse (WT) into the right knee joint, the width ofbilateral knee joints was measured and the difference in the width wasused as an indication for knee joint swelling to evaluate progress (FIG.6A) and the area under the curve (AUC) (FIG. 6B). The CNP transgenicmouse tended to have weak swelling in the right knee joint, and had asignificantly smaller AUC than that in the wild type. **: p<0.01, N.S.:not significant. Unpaired Student's t-test.

FIG. 7 shows histological changes in the right knee joint synovialmembrane in a collagenase OA model. It is a histological image of theright knee joint synovial membrane 28 days after administration of 3%collagenase physiological saline to a CNP transgenic mouse and awild-type mouse into the right knee joint. When administered 3%collagenase, the wild type mouse showed hyperplasia of synovialepithelial cells, granulation and inflammatory cell infiltration (FIG.7B). On the other hand, these findings were very few in the CNPtransgenic mice (FIG. 7C). FIG. 7A is a view of normal synovial tissue.

FIG. 8 shows histological changes in the articular cartilage of theright medial femoral condyle in a collagenase OA model. It is ahistological image of the right medial femoral condyle 28 days afteradministration of 3% collagenase physiological saline into the rightknee joint of a CNP transgenic mouse and a wild-type mouse. Thesafranine O stainability of the cartilage matrix decreased showing thedecreased proteoglycan content in the wild-type mouse (FIG. 8B), whilethe safranine O stainability was retained in the CNP transgenic mouse(FIG. 8C). FIG. 8A is a view of normal articular cartilage.

FIG. 9 is a graph showing the effectiveness in inhibiting articularswelling in a CNP collagenase OA mouse model receiving infusion. Thegraph shows the swelling of the right knee joint as measured 6 daysfollowing administration of 1.5% collagenase containing physiologicalsaline into the right knee joint of a C57BL/6 J Jcl mouse receivingcontinuous subcutaneous administration of CNP-22. CNP-22, both at 60 and600 ng/day, significantly inhibited the swelling of the right knee jointas compared to the solvent control group (vehicle). Unpaired Student'st-test.

FIG. 10 is a graph showing the effectiveness of CNP infusion ininhibiting articular swelling in a surgical OA mouse model. A C57BL/6 JJcl mouse was given continuous subcutaneous administration of CNP-22 andsubjected to the surgical procedures of anterocrucial ligament excision,tibial collateral ligament excision and medial meniscus total resectionin the right knee joint to induce osteoarthritis. The width of the rightand left knee joints was measured 4, 8 and 11 days postoperatively, andthe AUC of the difference was shown. CNP-22, both at 60 and 600 ng/day,significantly inhibited the swelling of the right knee joint compared tothe solvent control group (vehicle). Unpaired Student's t-test.

FIG. 11 is a graph showing the inhibitory effect of CNP-22 (6 ng/day,continuous subcutaneous administration), indomethacin (Indo., 1 mg/kg,oral administration) and a combination thereof on the knee jointswelling in a C57BL/6 J Jcl collagenase OA mouse model. FIG. 11A showsthe changes in swelling of the right knee joint over seven days afteradministration of 0.15% and 1.5% collagenase physiological saline intothe mouse right knee joint. FIG. 11B shows the area under the curve(AUC) of the graph in FIG. 11A. When the AUC was compared, CNP-22significantly inhibited the swelling of the right knee joint compared tothe solvent control group (vehicle) while indomethacin did not. On theother hand, a combination of CNP-22 and indomethacin showed remarkableinhibition, which was significantly stronger than CNP-22 used alone.Unpaired Student's t-test. *: p<0.05 (vs. vehicle), **: p<0.01 (vs.vehicle).

FIG. 12 is a graph showing the effectiveness of CNP-22 for arthritis inthe limb ends and body weight change in an adjuvant arthritis rat model.FIG. 12A represents changes in the arthritis score of the limb ends, andit indicates lower arthritis scores for the CNP-22 group. FIG. 12Brepresents body weight changes, and it indicates that on days 7 and 10from antigen sensitization, the CNP-22 group showed significantly heavybody weights compared to the solvent control group (vehicle). UnpairedStudent's t-test. *: p<0.05 (vs. vehicle).

FIG. 13 is a graph showing the effect of CNP-22 on the body weight in acollagen arthritis rat model. It indicates that while on days 21, 24 and28 from antigen sensitization, the solvent control group (vehicle)showed a significantly light body weight compared to the normal group,the CNP-22 group (CNP 6 μg/day) showed a significantly heavy body weightcompared to the solvent control group. Unpaired Student's t-test. ##:p<0.01 (vs. normal), *: p<0.05 (vs. vehicle).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is further described with reference to thefigures.

We analyzed the genotype of a CNP-transgenic mouse (CNP Tgm) produced asdescribed later in Example 2 using Southern blotting. As a result, wedetected 3 signals (“Wild type CNP gene”) in the wild type mouse, whiledetecting 2 signals (“Transgene”) derived from the transgene in the CNPTgm in addition to the wild-type CNP gene, as shown in FIG. 3. The CNPlevels in the liver, an organ expected to highly express said transgene,and in blood plasma were determined in order to study the expression ofCNP in the CNP Tgm. As a result, it was found that the CNP Tgm showedabout 10 fold and about 24 fold higher CNP levels in the liver and bloodplasma, respectively, than the wild type, demonstrating statisticallysignificant overexpression of CNP peptides (Table 1 in Example 4).

Furthermore, the thickness of the articular cartilage and the number ofchondrocytes were examined histologically to carry out a histologicalanalysis of the CNPTgm's articular cartilage, and the results indicatedthat the articular cartilage was statistically significantly thick (FIG.4) and the number of articular chondrocytes was statisticallysignificantly large (FIG. 5) in the CNPTgm. These results showed thatGC-B activators such as CNP may increase the thickness of the articularcartilage by increasing the number of chondrocytes.

In Example 6 described later, an osteoarthritic animal model was createdby injecting collagenase into the knee joint of a mouse to destabilizethe knee joint ligament and meniscus and inducing osteoarthritis (Am. J.Pathol. 1989; 135:1001-14). Osteoarthritic animal models derived fromthe CNPTgm and a normal mouse were used to evaluate the CNPTgm'sresistance to arthritis and articular cartilage degeneration. In theanimal model derived from the CNPTgm, when compared to the animal modelderived from a normal mouse, the knee joint swelling was significantlymilder, articular cartilage degeneration was inhibited to asignificantly larger degree, the changes in synovial cell growth,granulation and inflammatory cell infiltration in the synovial membranewere quite slight, and there was almost no change in the proteoglycancontent in the articular cartilage (FIGS. 6-8). These results indicatedthat GC-B activators have inhibitory effect on arthritis anddegeneration of the articular cartilage in osteoarthritis.

Furthermore, an osteoarthritic model was created using a normal mousetransplanted with an osmotic pump to examine the therapeutic effect ofCNP infusion on the osteoarthritic model. In the CNP group, the animalswere found to be resistant to knee joint swelling, have significantlyreduced degeneration of the articular cartilage, and show quite mildchanges in synovial cell growth, granulation and inflammatory cellinfiltration in the synovial membrane (FIG. 9). These results indicatedthat GC-B activators have a therapeutic effect on osteoarthritis.

Furthermore, a normal mouse transplanted with an osmotic pump wassubjected to the surgical procedures of anterocrucial ligament cut,tibial collateral ligament cut and medial meniscus total resection inthe right knee joint to induce osteoarthritis, and the therapeuticeffect of CNP infusion on the osteoarthritic model was examined. Resultsshowed that the AUC (area under the curve) was significantly lower inthe CNP group at either dose compared to the solvent control group (FIG.10). The results indicated that GC-B activators are also effective ininhibiting arthritis in osteoarthritis induced by physical overloadresulting from surgical procedures.

Furthermore, when CNP was administered to a collagenase OA mouse model,either alone or in combination with a nonsteroidal anti-inflammatorydrug (NSAID), CNP used alone significantly inhibited knee joint swellingwhile NSAID used alone did not, and the combination of CNP and NSAIDshowed an even stronger synergistic anti-swelling effect (Example 9,FIG. 11).

Furthermore, when the effect of CNP was further examined using adjuvantarthritis and collagen arthritis models generally used in laboratoriesas a rheumatoid arthritis (RA) model (Arthritis & Rheumatism,27:797-806, 1984; British Journal of Rheumatology, 33:798-807, 1994),the CNP group (rat) showed significantly reduced arthritis and a largerweight gain compared to control, indicating a significant improvement ingeneral condition (Examples 10 and 11 and FIGS. 12 and 13). Theseresults show the effectiveness of CNP for arthritis in rheumatoidarthritis.

From these demonstrative examples, we have now found that, without beingrestricted by any particular theory or experiment, GC-B activators suchas CNP possess anti-arthritis effect as well as assimilating action onthe articular cartilage.

Thus, the present invention provides a therapeutic or prophylactic agentfor arthritis comprising a GC-B activator as an active ingredient.

Examples of arthritis that can be treated or prevented according to thepresent invention include, but not limited to, those involving articularcartilage in particular, such as arthritis associated withosteoarthritis, synovitis, rheumatoid arthritis (rheumatoid arthritis(adults) and juvenile rheumatoid arthritis (children)), osteoarthritis,systemic lupus erythematodes (SLE), gout, scleroderma, psoriasis(psoriatic arthritis), mycotic infection such as blastomycosis,ankylosing spondilitis, Reiter's syndrome, septic arthritis, adult Stilldisease, tertiary Lyme disease (late stage), tuberculosis (tuberculousarthritis), viral infection (viral arthritis), and arthritis caused byinfection with gonorrhea (gonococcal arthritis) and bacteria(non-gonococcal bacterial arthritis).

In one embodiment of the present invention, a preferred arthritis isosteoarthritis, or arthritis associated with osteoarthritis.

Osteoarthritis is a disease caused by the degeneration and destructionof the articular cartilage, and examples of applicable osteoarthritisinclude, for example, (1) osteoarthritis of weight-bearing joints, suchas gonarthrosis in the knee joint, coxarthrosis in the hip joint, footosteoarthritis in the foot and spinal osteoarthritis in the spine, and(2) osteoarthritis of non-weight-bearing joints, such as shoulderosteoarthritis in the shoulder, elbow osteoarthritis in the elbow, handosteoarthritis in the hand (for example, Heberden's nodes, Bouchard'snodes, thumb CM osteoarthritis) and temporomandibular arthrosis in thejaw.

In one embodiment of the present invention, the osteoarthritis isosteoarthritis affecting weight-bearing joints, preferably gonarthrosisor coxarthrosis.

In another embodiment of the present invention, the osteoarthritis isosteoarthritis affecting non-weight-bearing joints, preferablytemporomandibular arthrosis.

Therapeutic or prophylactic agents of the present invention can also beapplied in the treatment or prevention of rheumatoid arthritis.Rheumatoid arthritis is thought to be an autoimmune disease, andalthough it has different etiology from osteoarthritis, it involves, aswith osteoarthritis, the degeneration of the articular cartilagesurfaces and the destruction of cartilage as it progresses.Consequently, therapeutic agents of the present invention may beadministered to inhibit or relieve arthritis.

The terms “treatment,” “method for treating” and “therapeutic agent,” asused herein, mean eliminating, inhibiting or relieving the symptoms of apatient with arthritis according to the present invention, or methods ordrugs for that purpose. In addition, the terms “prevention” and“prophylactic agent” mean preventing arthritis or drugs for thatpurpose.

As used in the invention, the term “guanyl cyclase B (GC-B)” has thesame meaning as natriuretic peptide receptor B (NPR-B).

As used in the invention, the term “activity of GC-B” has the samemeaning as guanyl cyclase activity. In the present invention, a guanylcyclase B (GC-B) activator or GC-B activator is a peptide or anonpeptidic low-molecular-weight compound, preferably a CNP peptide or aderivative thereof, that can bind to and activate GC-B, which is knownas a CNP receptor. Peptides as used herein refer to a substanceconsisting of amide bond linkages of a plurality of (L-, D- and/ormodified) amino acids, and include polypeptides and proteins. A GC-Bactivator can be identified, for example, by expressing a GC-B receptorin a cultured cell line such as COS-7, adding a candidate agent to themedium, culturing the cell line for a certain time period at a certaintemperature (for example, 37° C., 5 minutes), and measuring the amountof intracellular cGMP produced (Science 1991, 252: 120-123). Using suchan assay system, and using the amount of intracellular cGMP productionas an indication, a GC-B activator may be identified and used in thepresent invention.

According to one embodiment of the invention, the GC-B activator is apeptide, and preferably CNP or a derivative thereof. Preferred CNP isselected from CNP-22 and CNP-53 from mammals, including human, or birds,and more preferably CNP-22 of SEQ ID NO: 1 or CNP-53 of SEQ ID NO: 2.

According to another embodiment of the invention, the CNP derivative asdescribed above has a deletion, substitution or addition of one orseveral amino acids in the amino acid sequence of SEQ ID NO: 1 or SEQ IDNO: 2, while possessing a CNP activity. Alternatively, the CNPderivative comprises a sequence having about 70% or more, about 80% ormore, about 85% or more, about 90% or more, about 95% or more, about 97%or more, about 98% or more, or about 99% or more identity with the aminoacid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and retains CNP activity.

The term “one or several” as used herein generally represents anyinteger between 1 and 10, preferably between 1 and 5, more preferablybetween 1 and 3. The “% identity” between two amino acid sequences maybe determined using techniques well known to those skilled in the art,such as BLAST protein search (Altschul, S. F., Gish, W., Miller, W.,Myers, E. W. & Lipman, D. J. (1990) “Basic Local Alignment Search Tool”J. Mol. Biol. 215:403-410).

Examples of CNPs usable in the present invention include CNPs frommammals including human (CNP-22: Biochem. Biophys. Res. Commun. 1990;168: 863-870, International Publication No. WO 91/16342, CNP-53:Biochem. Biophys. Res. Commun. 1990; 170:973-979, Japanese PatentPublication (Kokai) No. 4-74198A (1992), Japanese Patent Publication(Kokai) No. 4-139199A (1992), Japanese Patent Publication (Kokai) No.4-121190A (1992)), CNPs from birds (Japanese Patent Publication (Kokai)No. 4-120094A (1992)), CNPs from amphibians (Japanese Patent Publication(Kokai) No. 4-120095A (1992)), and CNP derivatives such as CNP analogouspeptides disclosed in Japanese Patent Publication (Kokai) No. 6-9688A(1994) and International Publication No. WO 02/074234.

CNP-22 and CNP-53, which consist of 22 and 53 amino residuesrespectively, are known as naturally occurring CNPs. Because CNPs have ahigh homology in their sequences between birds and mammals includinghuman, i.e. regardless of the kind of animals, CNPs from birds andmammals including human, preferably CNPs from mammals including human,and more preferably CNPs from human, can be used in the presentinvention. The amino acid sequence of human CNP-22 or CNP-53 has thesequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 respectively, representedby:

Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met SerGly Leu Gly Cys (human CNP-22; SEQ ID NO: 1); or

Asp Leu Arg Val Asp Thr Lys Ser Arg Ala Ala Trp Ala Arg Leu Leu Gln GluH is Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys GlyCys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys(human CNP-53; SEQ ID NO: 2),

each of which has an intramolecular disulfide bond, i.e. between 6-Cysand 22-Cys in human CNP-22 or between 37-Cys and 53-Cys in human CNP-53,forming a cyclic peptide structure.

Pig CNP-22 and rat CNP-22 have the same amino acid sequence as humanCNP-22, whereas the amino acid residues at positions 17 and 28 are Hisand Gly, respectively, in pig CNP-53 and rat CNP-53, and they are Glnand Ala in human CNP-53, i.e., two amino acids are different in CNP-53between human and pig or rat (Japanese Patent Publication (Kokai) No.4-139199A (1992), Japanese Patent Publication (Kokai) No. 4-121190A(1992), and Japanese Patent Publication (Kokai) No. 4-74198A (1992)). Inaddition, chicken CNP-22 has the same primary structure as human CNP-22,with the exception that the amino acid residue at position 9 is Val(Japanese Patent Publication (Kokai) No. 4-120094A (1992)).

The CNPs usable in the invention include CNPs purified from naturalsources, recombinant CNPs produced by known genetic engineeringtechniques, and CNPs produced by known chemical syntheses (for example,a solid phase synthesis using peptide synthesizer), preferably humanCNP-22 and human CNP-53 produced by genetic engineering techniques.Production of human CNPs by genetic engineering techniques comprises,for example, the steps of incorporating the DNA sequence of human CNP-22or CNP-53 (Japanese Patent Publication No. 4-139199A (1992)) into avector such as plasmid or phage, transforming the vector into aprocaryotic or eucaryotic host cell, such as E. coli or yeast, andexpressing the DNA in suitable culture medium, preferably allowing thecells to secrete the CNP peptide extracellularly, and collecting andpurifying the CNP peptide produced. Polymerase chain reaction (PCR)technique can also be used to amplify target DNA.

Basic techniques such as genetic recombination, site-directedmutagenesis and PCR techniques are well-known to those skilled in theart, which are described, for example, in J. Sambrook et al., MolecularCloning, A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press (1990); Ausubel et al., Current Protocols In MolecularBiology, John Wiley & Sons (1998), and said techniques as disclosedtherein may be used for the present invention. As the vectors,commercially available vectors or vectors as disclosed in publicationsmay also be used.

CNP derivatives that may be used in the present invention have the CNPactivity and have a cyclic peptide structure having a disulfide bondbetween two cysteine residues as seen in human CNP-22 or CNP-53.Examples of the CNP derivatives include: fragments of the CNPs asdescribed above; peptides having a substitution of at least one aminoacid by another amino acid in the CNPs above or fragments thereof;peptides having a deletion of at least one amino acid in the CNPs aboveor partial peptides thereof; and peptides having an addition of at leastone amino acid in the CNPs above or partial peptides thereof. As usedherein, the substitution, deletion or addition of amino acids means thata certain number of amino acids are substituted, deleted or added by awell-known method such as site-directed mutagenesis, with the provisothat the CNP activity is not lost. For example, the CNP-22 or CNP-53derivatives have a substitution, deletion or addition of one or severalamino acids in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2,possessing the CNP activity.

In general, the substitution of amino acids is preferably a substitutionbetween conservative amino acids. Conservative amino acids may beclassified according to, for example, polarity (or hydrophobicity) ortypes of electric charges. Examples of nonpolar, uncharged amino acidsinclude glycine, alanine, valine, leucine, isoleucine, proline, etc.;aromatic amino acids include phenylalanine, tyrosine and tryptophan;polar, uncharged amino acids include serine, threonine, cysteine,methionine, asparagine, glutamine, etc.; negatively charged amino acidsinclude aspartic acid and glutamic acid; and positively charged aminoacids include lysine, arginine and histidine.

In the present invention the CNP activity refers to the activity to acton GC-B to increase guanyl cyclase activity, the activity to eliminate,inhibit or relieve arthritis including osteoarthritis, and the activityto promote the growth of the articular cartilage. The CNP activity canbe determined by measuring cellular guanyl cyclase activity, such as,for example, intracellular production of cGMP, and/or by administering aCNP or a derivative thereof for a certain period to mouse or rat modelsof arthritis, osteoarthritis or rheumatoid arthritis, and measuring asdescribed later in Examples 7 to 10 the effectiveness in inhibiting thearthritis or the degeneration of the articular cartilage.

Examples of CNP-22 analogous peptides include the following cyclicpeptides described in Japanese Patent Publication (Kokai) No. 6-9688(1994) and International Publication No. WO02/074234 (where underlinesin the sequences represent variations from human CNP-22). Gly Leu SerLys Gly Cys Phe Gly Leu (SEQ ID NO:3) Lys Leu Asp Arg Ile Gly Ala MetSer Gly Leu Gly Cys Gly Leu Ser Lys Gly Cys Phe Gly Leu (SEQ ID NO:4)Lys Leu Asp Arg Ile Gly Ser Gln Ser Gly Leu Gly Cys Gly Leu Ser Lys GlyCys Phe Gly Leu (SEQ ID NO:5) Lys Leu Asp Arg Ile Gly Ser Ala Ser GlyLeu Gly Cys Cys Phe Gly Leu Lys Leu Asp Arg Ile (SEQ ID NO:6) Gly SerMet Ser Gly Leu Gly Cys Ser Leu Arg Arg Ser Ser Cys Phe Gly (SEQ IDNO:7) Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys Gly LeuSer Lys Gly Cys Phe Gly Leu (SEQ ID NO:8) Lys Leu Asp Arg Ile Gly SerMet Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr Cys Phe Gly Leu Lys Leu AspArg Ile (SEQ ID NO:9) Gly Ser Gln Ser Gly Leu Gly Cys AsnSer Phe Arg Tyr Cys Phe Gly Xaa Xbb XccAsp Arg Ile (SEQ ID NO:10) GlyXdd Xee Ser Xff Xgg Gly Cys (wherein Xaa = Leu, Ile, Val; Xbb = Lys,Leu, Met; Xcc = Leu, Ile, Ala, Val; Xdd = Ser, Ala, Gly, Thr, Asn; Xee =Met, Ala, Trp, His, Lys, Ser, Gly; Xff = Gly, Lys, Ala, Leu; Xgg = Leu,met).

In addition, CNP-53 analogous peptides include cyclic peptidescontaining similar variations of amino acids corresponding to the CNP-22analogous peptides described above.

The present invention also provides an agent for promoting the growth ofarticular chondrocyte, comprising a GC-B activator as an activeingredient. This invention is based on the action of a GC-B activator toincrease articular chondrocytes. Examples of the GC-B activator are theCNPs as defined above, or derivatives thereof. The CNP is preferablyCNP-22 or CNP-53 from mammals, including human, or birds, morepreferably CNP-22 of SEQ ID NO:1 or CNP-53 of SEQ ID NO:2. The CNPderivative has deletion, substitution or addition of one or severalamino acids in the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2,and has CNP activity. Other GC-B activators can be identified, forexample, by expressing a GC-B receptor in cultured cells such as COS-7,adding a candidate agent to the medium, culturing the cells for acertain time period at a certain temperature (for example, 37° C., 5minutes), and measuring the amount of intracellular cGMP produced(Science 1991; 252: 120-123). Using such an assay system and using theamount of produced intracellular cGMP as an indication, a GC-B activatormay be identified and used for the present invention.

The present invention also provides a method for inhibiting arthritis,wherein the arthritis is inhibited by activating GC-B. The presentinvention also provides a method for promoting the growth of articularchondrocyte comprising promoting the growth by activating GC-B. Theseinventions are based on the finding that the arthritis as defined above,preferably osteoarthritis, can be inhibited and the growth of articularchondrocytes can be enhanced by using a GC-B activator, or by activatingGC-B. In one embodiment of the present invention, the GC-B is activatedby the CNPs as defined above or derivatives thereof.

The present invention further provides a method for screening an agentfor promoting the growth of articular chondrocyte, comprising screeningcandidate agents for the ability to promote the growth of articularchondrocyte using the GC-B activity as an indication. Because GC-B isknown to catalyze the synthesis of the second messenger cGMP from GTPthrough guanyl cyclase activity, the GC-B activity may be determined asthe amount of intracellular cGMP produced.

According to an embodiment of the present invention, the screeningmethod as described above may include the steps of preparing cellsexpressing GC-B, or cells derived from articular chondrocytes, culturingthe cells in the presence of a candidate agent, and screening thecandidate agent for the ability to promote the growth of articularchondrocyte using the cellular guanyl cyclase activity, for example theamount of intracellular cGMP produced, as an indication.

According to a preferred embodiment of the present invention, thescreening method comprises preparing a cultured cell line that had beenforced to express GC-B, culturing the cell line in the presence orabsence of a candidate agent, determining the amount of intracellularcGMP produced, and screening the candidate agent for the ability topromote the growth of articular chondrocyte using as an indication thedifference between the amounts of intracellular cGMP produced in thepresence and absence of the candidate agent.

The screening method according to the invention may be used to screen anarticular chondrocyte growth promoter by, for example, expressing GC-Bin cultured cells such as COS-7, adding a candidate agent to the medium,culturing the cells for a certain time period at a certain temperature(for example, 37° C., 5 minutes), and determining the amount ofintracellular cGMP produced (Science 1991; 252: 120-123).

The present invention further provides a method for screening atherapeutic agent for osteoarthritis, rheumatoid arthritis or otherarthritis comprising screening candidate agents for an agent capable oftreating osteoarthritis, rheumatoid arthritis or other arthritis usingGC-B activity as an indication. As described above, the GC-B activitycan be determined as guanyl cyclase activity, for example the amount ofintracellular cGMP produced.

In one embodiment of the present invention, the screening method asdescribed above may include the steps of: preparing cells that expressGC-B, or cells from articular chondrocytes; culturing the cells in thepresence of a candidate agent; and screening the candidate agents for anagent capable of treating osteoarthritis, rheumatoid arthritis or otherarthritis using the cellular guanyl cyclase activity, for example theamount of intracellular cGMP produced, as an indication.

According to a preferred embodiment of the present invention, thescreening method comprises preparing a cultured cell line that had beenforced to express GC-B, culturing the cell line in the presence orabsence of a candidate agent, determining the amount of intracellularcGMP produced, and screening the candidate agents for an agent capableof treating osteoarthritis, rheumatoid arthritis or arthritis using asan indication the difference between the amounts of intracellular cGMPproduced in the presence and absence of the candidate agent.

The screening method according to the invention may be used to screen atherapeutic agent for osteoarthritis, rheumatoid arthritis or otherarthritis by, for example, expressing GC-B in cultured cells such asCOS-7, adding a candidate agent to the medium, culturing the cells for acertain time period at a certain temperature (for example, 37° C., 5minutes), and measuring the amount of intracellular cGMP produced(Science 1991; 252: 120-123).

The therapeutic or prophylactic agent of the present invention forarthritis, such as osteoarthritis, is formulated into preparations fororal or parenteral administration by combining the GC-B activatordefined above as an active ingredient with a pharmaceutically acceptablecarrier, excipient, additive, or the like.

Examples of the carriers and excipients for preparation include lactose,magnesium stearate, starch, talc, gelatin, agar, pectin, gum arabic,olive oil, sesame oil, cacao butter, ethylene glycol, and othersconventionally used.

Examples of solid compositions for oral administration include tablets,pills, capsules, powders, granules, and the like. In such solidcompositions, at least one active ingredient is mixed with at least oneinert diluent, such as lactose, mannitol, glucose,hydroxypropylcellulose, microcrystal cellulose, starch,polyvinylpyrrolidone, magnesium aluminometasilicate, or the like. Thecomposition may, according to a conventional method, also containadditives other than inert diluents, for example, a lubricant such asmagnesium stearate, a disintegrating agent such as fibrous calciumglycolate, and a dissolution auxiliary agent such as glutamic acid oraspartic acid. Tablets or pills may, as required, be coated with aglycocalyx, such as sucrose, gelatin or hydroxypropyl methylcellulosephthalate, or with a gastro- or enteric-film, or with two or morelayers. Capsules of an absorbable material, such as gelatine, are alsoincluded.

Liquid compositions for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs, andmay also contain conventional inert diluents, such as purified water andethanol. The composition may contain, other than the inert diluent, anadjuvant, such as wetting and suspending agents, a sweetening agent, aflavor, an aromatic, and a preservative.

Examples of parenteral injections include sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of aqueous solutions andsuspensions include water for injection and physiological saline forinjection. Examples of non-aqueous solutions and suspensions includepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,alcohols such as ethanol, and polysorbate 80®. These compositions mayfurther contain adjuvants, such as preservatives, wetting agents,emulsifiers, dispersants, stabilizers (for example, lactose), anddissolution auxiliary agents (e.g., glutamic acid and aspartic acid).The above-described materials may be sterilized by conventionalsterilization methods, such as filter sterilization with amicrofiltration membrane, heat sterilization such as autoclaving, orincorporation of disinfectants. Injections may be liquid preparations,or freeze-dried preparations that may be reconstituted before use.Examples of excipients for freeze-drying include sugar alcohols andsugars, such as mannitol and glucose.

The therapeutic or prophylactic agent of the present invention isadministered by either oral or parenteral administration methodscommonly used for pharmaceuticals. Preferred are parenteraladministration methods, for example, injection (e.g., subcutaneous,intravenous, intramuscular and intraperitoneal injections), percutaneousadministration, trans-mucosal administration (e.g., transnasal andtransrectal), and trans-pulmonary administration. Oral administrationmay also be used.

The dosage of a GC-B activator, preferably a CNP as defined above or aderivative thereof, which is an active ingredient contained in thecomposition of the present invention, may be determined depending on thetype of disease to be treated, the severity of the disease, patient'sage, and the like, and may generally range from 0.005 μg/kg to 100mg/kg, preferably from 0.02 μg/kg to 5 mg/kg., more preferably from 0.02μg/kg to 0.25 mg/kg. However, the drugs containing CNPs according to thepresent invention are not limited to these dosages.

The therapeutic or prophylactic agent of the present invention may becombined with conventional or new therapeutic agents, such asanti-inflammatory drugs, hyaluronic acid and adrenocortical steroid, aswell as with orthopedic surgical operations, such as arthroscopicsurgery, artificial joint replacement and osteotomy.

Combination of an anti-inflammatory drug in particular, for example atleast one nonsteroidal anti-inflammatory drug, with a GC-B activator(for example, the CNPs as defined above or derivatives thereof) canprovide a synergistic inhibitory effect on arthritis (Example 10).

The “nonsteroidal anti-inflammatory drug” as used herein refers to ananti-inflammatory drug without steroid backbone, and those having theaction to inhibit cyclooxygenase enzymes involved in the production ofprostaglandins are preferred. Examples of nonsteroidal anti-inflammatorydrugs usable in the present invention include, but not limited to,indomethacin (for example, Indacin™), ibuprofen (for example, Brufen™),piroxicam, salicylic acid, diclofenac (for example, Voltaren™),ketoprofen, naproxen, and piroxicam.

Furthermore, the synergistic effect of a combination of the GC-Bactivator described above with a nonsteroidal anti-inflammatory drugmeans that, compared to when the GC-B activator is used alone, theactivation of GC-B is enhanced, or in other words, the active ingredientof the nonsteroidal anti-inflammatory drug described above serves as anactivation promoter in activating GC-B with the GC-B activator.

Thus, the present invention further provides an activation promoter fora GC-B activator, comprising a nonsteroidal activator.

GC-B activators include the CNPs or derivatives thereof as definedabove. Examples of CNPs are CNP-22 and CNP-53 from mammals, includinghuman, or birds, more specifically CNP-22 of SEQ ID NO:1 or CNP-53 ofSEQ ID NO:2. Examples of the CNP derivatives include those having adeletion, substitution or addition of one or several amino acids in theamino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, and retaining CNPactivity.

In the present invention, the nonsteroidal activator as described aboveis preferably a cyclooxygenase inhibitor. Examples of the cyclooxygenaseinhibitor include, but not limited to, indomethacin, ibuprofen,piroxicam, salicylic acid, diclofenac, ketoprofen, naproxen andpiroxicam.

The above descriptions of the dosage form, dosage and administrationmethod of the therapeutic and prophylactic agents of the presentinvention may be applied as is to the activation promoter of the presentinvention.

The present invention further provides a method for activating a GC-Bactivator, wherein the activation promoter as described above is used.

The activation promoter and method of the present invention as describedabove may be used, for example, for treating diseases, such asarthritis, effectively in patient through GC-B activation.

The invention includes, but not limited to, the following items.

(1) A therapeutic or prophylactic agent for arthritis comprising aguanyl cyclase B (GC-B) activator as an active ingredient.

(2) The therapeutic or prophylactic agent according to (1) above,wherein the arthritis is osteoarthritis.

(3) The therapeutic or prophylactic agent according to (2) above,wherein the osteoarthritis is osteoarthritis of weight-bearing ornon-weight-bearing joints.

(4) The therapeutic or prophylactic agent according to (3) above,wherein the osteoarthritis is degenerative gonarthrosis.

(5) The therapeutic or prophylactic agent according to (3) above,wherein the osteoarthritis is degenerative coxarthrosis.

(6) The therapeutic or prophylactic agent according to (3) above,wherein the osteoarthritis is temporomandibular arthrosis.

(7) The therapeutic or prophylactic agent according to (I) above,wherein the arthritis is caused by rheumatoid arthritis.

(8) The therapeutic or prophylactic agent according to (1) above,wherein the arthritis is caused by osteoarthritis.

(9) The therapeutic or prophylactic agent according to any of items (1)to (8) above, wherein the GC-B activator is a type C natriuretic peptide(CNP) or a derivative thereof

(10) The therapeutic or prophylactic agent according to (9) above,wherein the CNP is selected from CNP-22 and CNP-53 derived from mammals,including human, or birds.

(11) The therapeutic or prophylactic agent according to (9) above,wherein the CNP is CNP-22 of SEQ ID NO:1 or CNP-53 of SEQ ID NO:2.

(12) The therapeutic or prophylactic agent according to (9) above,wherein the derivative has deletion, substitution or addition of one orseveral amino acids in the amino acid sequence of SEQ ID NO:1 or SEQ IDNO:2, and has CNP activity.

(13) The therapeutic or prophylactic agent according to any of items (1)to (12) above, further comprising at least one nonsteroidalanti-inflammatory drug.

(14) An agent for promoting the growth of articular chondrocytecomprising a GC-B activator as an active ingredient.

(15) The agent according to (14) above, wherein the GC-B activator is aCNP or a derivative thereof.

(16) The agent according to (15) above, wherein the CNP is CNP-22 orCNP-53 from mammals, including humans, or birds.

(17) The agent according to (15) above, wherein the CNP is CNP-22 of SEQID NO:1 or CNP-53 of SEQ ID NO:2.

(18) The agent according to (15) above, wherein the derivative has adeletion, substitution or addition of one or several amino acids in theamino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, while possessing aCNP activity.

(19) The agent according to any of items (14) to (18) above, furthercomprising at least one nonsteroidal anti-inflammatory drug.

(20) A method for inhibiting arthritis, wherein the arthritis isinhibited by activating GC-B.

(21) The method for inhibition according to (20) above, wherein the GC-Bis activated by a CNP or a derivative thereof

(22) The method for inhibition according to (21) above, wherein the CNPis CNP-22 or CNP-53 from mammals, including human, or birds.

(23) The method for inhibition according to (21) above, wherein the CNPis CNP-22 of SEQ ID NO:1 or CNP-53 of SEQ ID NO:2.

(24) The method for inhibition according to (21) above, wherein thederivative has a deletion, substitution or addition of one or severalamino acids in the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2,while possessing a CNP activity.

(25) The method for inhibition according to any of items (20) to (24)above, wherein the GC-B is activated by a combination of a CNP or aderivative thereof and at least one nonsteroidal anti-inflammatory drug.

(26) A method for promoting the growth of articular chondrocyte, whereinthe articular chondrocyte growth is accelerated by activating GC-B.

(27) The method according to (26) above, wherein the GC-B is activatedby a CNP or a derivative thereof

(28) The method according to (27) above, wherein the CNP is CNP-22 orCNP-53 derived from mammals, including human, or birds.

(29) The method according to (27) above, wherein the CNP is CNP-22 ofSEQ ID NO:1 or CNP-53 of SEQ ID NO:2.

(30) The method according to (27) above, wherein the derivative has adeletion, substitution or addition of one or several amino acids in theamino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, while possessing aCNP activity.

(31) The method according to any of items (26) to (30) above, whereinthe GC-B is activated by a combination of a CNP or a derivative thereofand at least one nonsteroidal anti-inflammatory drug.

(32) A method for screening an articular chondrocyte growth promotercomprising screening a candidate agent for the ability to promotearticular chondrocyte using GC-B activity as an indication.

(33) The method according to (32) above, comprising preparing culturedcells that express GC-B, or cells from articular chondrocytes, culturingthe cells in the presence of a candidate agent, and screening thecandidate agents for the ability to promote the growth of articularchondrocyte using the cell's GC-B activity as an indication.

(34) The method according to (32) or (33) above, wherein the GC-Bactivity is determined as an amount of intracellular cGMP produced.

(35) The method according to any of items (32) to (34) above, comprisingpreparing a cultured cell line that had been forced to express GC-B,culturing the cell line in the presence or absence of a candidate agent,determining the amount of intracellular cGMP produced, and screening thecandidate agents for the ability to accelerate articular chondrocytegrowth using, as an indication, the difference between the amounts ofintracellular cGMP produced in the presence and absence of the candidateagent.

(36) A method for screening a therapeutic agent for osteoarthritis,rheumatoid arthritis or other arthritis comprising screening candidateagents for an agent capable of treating osteoarthritis, rheumatoidarthritis or other arthritis using GC-B activity as an indication.

(37) The method according to (36) above, comprising preparing culturedcells that express GC-B, or cells from articular chondrocytes,incubating the cells in the presence of a candidate agent, and screeningthe candidate agents for an agent capable of treating osteoarthritis,rheumatoid arthritis or other arthritis using the cellular GC-B activityas an indication.

(38) The method according to (36) or (37) above, wherein the GC-Bactivity is determined as an amount of intracellular cGMP produced.

(39) The method according to any of items (36) to (38) above, comprisingpreparing a cultured cell line that had been forced to express GC-B,culturing the cell line in the presence or absence of a candidate agent,determining the amount of intracellular cGMP produced, and screening thecandidate agents for an agent capable of treating osteoarthritis,rheumatoid arthritis or other arthritis using as an indication thedifference between the amounts of intracellular cGMP produced in thepresence and absence of the candidate agent.

(40) A therapeutic or prophylactic agent for osteoarthritis comprising aguanyl cyclase B (GC-B) activator as an active ingredient.

(41) The therapeutic or prophylactic agent for osteoarthritis accordingto (40) above, further comprising at least one nonsteroidalanti-inflammatory drug.

(42) A therapeutic or prophylactic agent for rheumatoid arthritiscomprising a guanyl cyclase B (GC-B) activator as an active ingredient.

(43) The therapeutic or prophylactic agent for rheumatoid arthritisaccording to (42) above, further comprising at least one nonsteroidalanti-inflammatory drug.

(44) A method for treating arthritis comprising administering a GC-Bactivator to a patient in need of treatment for the arthritis.

(45) The method according to (44) above, wherein the GC-B activator is aCNP or a derivative thereof

(46) The method according to (44) or (45) above, wherein the arthritisis osteoarthritis.

(47) The method according to (46) above, wherein the osteoarthritis isosteoarthritis of weight-bearing or non-weight-bearing joints.

(48) The method according to (47) above, wherein the osteoarthritis isdegenerative gonarthrosis, degenerative coxarthrosis, ortemporomandibular arthrosis.

(49) The method according to (44) above, wherein the arthritis is causedby rheumatoid arthritis.

(50) The method according to (44) above, wherein the arthritis is causedby osteoarthritis.

(51) The method according to any of items (45) to (50) above, whereinthe CNP is selected from CNP-22 and CNP-53 derived from mammals,including humans, or birds.

(52) The method according to any of items (45) to (50) above, whereinthe CNP is CNP-22 of SEQ ID NO:1 or CNP-53 of SEQ ID NO:2.

(53) The method according to any of items (45) to (50) above, whereinthe derivative has a deletion, substitution or addition of one orseveral amino acids in the amino acid sequence of SEQ ID NO:1 or SEQ IDNO:2, while possessing a CNP activity.

(54) The method according to any of items (44) to (53) above, whereinthe GC-B activator is contained in combination with at least onenonsteroidal anti-inflammatory drug.

(55) An activation promoter for a GC-B activator, comprising anonsteroidal activator.

(56) The activation promoter according to (55) above, wherein the GC-Bactivator is a CNP or a derivative thereof.

(57) The activation promoter according to (56) above, wherein the CNP isselected from CNP-22 and CNP-53 from mammals, including human, or birds.

(58) The activation promoter according to (56) above, wherein the CNP isCNP-22 of SEQ ID NO:1 or CNP-53 of SEQ ID NO:2.

(59) The activation promoter according to (56) above, wherein thederivative has a deletion, substitution or addition of one or severalamino acids in the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2,while possessing a CNP activity.

(60) The activation promoter according to (55) above, wherein thenonsteroidal activator is a cyclooxygenase inhibitor.

(61) The activation promoter according to (60) above, wherein thecyclooxygenase inhibitor is selected from the group consisting ofindomethacin, ibuprofen, piroxicam, salicylic acid, diclofenac,ketoprofen, naproxen and piroxicam.

(62) A method for activating a GC-B activator, wherein the activationpromoter according to any of (55) to (61) above is used.

The present invention will be described in more detail by the followingexamples, which are for illustrative purposes only and are not intendedto limit the scope of the invention. Thus, the present invention is notlimited to those examples.

EXAMPLES Example 1 Construction of Vector for Preparing CNP TransgenicMouse

As shown in FIG. 1A, the murine CNP cDNA (526 bp; FEBS Lett.276:209-213, 1990) was subcloned into pGEM-T easy vector (Promega), andwas then cut with Pst I and blunt-ended to prepare a mouse CNP cDNA. Thevector PSG 1 (Promega; FIG. 1B) was cut with EcoRI, blunt-ended andligated with the murine CNP cDNA, as shown in FIG. 1C, to prepare aSAP-mCNP vector (pSG1-CNP).

Example 2 Production of CNP Transgenic Mouse

A DNA fragment for injection was prepared as follows. The SAP-mCNPvector (pSG1-CNP; FIG. 1C) with an inserted CNP gene was first treatedwith Hind III and Xho I to cut out a fragment (about 2.3 kb) containingthe CNP gene. The fragment was then collected using Gel Extraction Kit(QIAGEN), and was diluted with PBS⁻ at a concentration of 3 ng/μl,thereby obtaining the DNA fragment for injection (FIG. 2).

The mouse egg at pronucleus stage into which the DNA fragment wasinjected was collected as follows. First, a C57BL/6 female mouse (CleaJapan, Inc.) was injected intraperitoneally with 5 i.u pregnant mareserum gonadotropin (PMSG), and 48 hours later, with 5 i.u humanchorionic gonadotropin (hCG), in order to induce superovulation. Thisfemale mouse was crossed with a congeneric male mouse. In the nextmorning of the crossing, in the female mouse the presence of a plug wasconfirmed and subsequently the oviduct was perfused to collect a mouseegg at pronucleus stage.

The DNA fragment for injection was injected into the pronucleus eggusing a micromanipulator (Latest Technology in Gene Targeting (Yodosha,Japan), 190-207, 2000). Specifically, the DNA fragment was injected into660 C57BL/6J embryos, and on the following day, 561 embryos at 2-cellstage were transplanted into the oviducts of recipient females on day 1of false pregnancy at about 10 per each side of the oviduct (about20/animal).

Recipient females, which had not been delivered of offsprings by theexpected date of delivery, were subjected to cesarean section, resultingin the birth of offsprings which were raised by a foster mother. Total136 offsprings were obtained, 5 of which were transgenic mice with anintroduced CNP gene (hereafter referred to as “Tgm”). Hereinafter, themouse initially obtained is referred to as the Founder.

All Founder mice were male, and the subsequent generation of offsprings(i.e., F1 mice) were obtained from four of the five lines.

Example 3 Genotype Analysis of CNP Transgenic Mouse

Genotype analysis was performed by Southern blotting according toprocedures as described below.

The tail (about 15 mm) was taken from the 3-week old mouse and treatedwith proteinase K (at 55° C., with shaking at 100 rpm over day andnight) to obtain a lysis solution. The obtained solution was thensubjected to an automated nucleic acid separator (KURABO NA-1000;Kurabo, Japan) to prepare genomic DNA. The genomic DNA (15 μg) wastreated with Pvu II (200 U), then with phenol-chloroform to remove therestriction enzyme, and was precipitated with ethanol to collect theDNA. The obtained DNA was dissolved in 25 μL of TE and subjected toelectrophoresis on 0.7% agarose gel (at 50V constant voltage), then thegel was treated with 0.25M HCl solution for 15 minutes to cleave theDNA, washed with water, and blotted overnight onto a nylon membrane in0.4M NaOH solution. Thereafter, the DNA on the membrane was fixed by theUV crosslink method. The membrane was treated (at 42° C. for 2 hours)with a hybridization solution (50% formamide, 0.5× Denhardt's, 0.5% SDS,5×SSPE), and a ³²P labeled probe, which has been prepared with BcaBESTLabeling Kit (TaKaRa, Japan) using the CNP cDNA (about 0.5 kb) as atemplate, was added to the membrane for hybridization at 42° C.overnight. After treatment with a detergent solution (2×SSC, 0.1% SDS)at 55° C. for 20 minutes, the membrane was exposed to an Imaging Plate(Fuji Film) overnight to detect signals of the transgene using BAS2000(Fuji Film, Japan) (FIG. 3). In the wild-type mouse (WT) 3 signals(wild-type CNP gene) were detected, while in the transgenic mouse (Tgm)2 signals (transgene) derived from the transgene were detected inaddition to the wild-type CNP gene.

Example 4 CNP Expression in CNP Transgenic Mouse

A CNP-22 EIA measuring kit (PHOENIX PHARMACEUTICALS INC.) was used forthe determination of a CNP level.

Three each of 7-week old male and female CNP transgenic mice, as well as3 each of male and female normal litter of mice, were euthanized byexsanguination from the postcava under ether anesthesia.

The liver, which is an organ expected to exhibit high expression of thetransgene, was removed, and the EIA assay buffer from the measuring kitas above was added at 1 ml per 0.1 g of liver weight, followed bycooling on ice. The liver was homogenized in a Waring blender(Physcotron), and after centrifugation (at 2,000 rpm for 5 minutes), thesupernatant was used as a sample for the determination of CNP-22 levels.

One mg of ethylenediaminetetraacetate-4Na (Junsei Chemical Co., Ltd.,Japan) and 2 trypsin-inhibition units of aprotinin (Sigma) were added tothe drawn blood and agitated to separate blood plasma, which was used asa sample for the determination of CNP-22 levels.

The results are shown in Table 1. TABLE 1 CNP expression in CNPtransgenic mouse Liver Plasma (ng/g tissue) mean ± SD (ng/mL) mean ± SDWild No.1 38.8 29.3 ± 20.5 0.3 0.3 ± 0.06 type No.2 5.9 0.4 No.3 43.30.3 CNP No.1 293.3   290 ± 81.7** 10.3 8.0 ± 4.7^(#) tgm No.2 370.0 11.1No.3 206.7 2.6**p < 0.01 (unpaired Student's t-test)^(#)p < 0.05 (Wilcoxon rank sum test)

The CNP transgenic mouse showed about 10 fold and about 24 fold higherCNP-22 level in the liver and blood plasma respectively, than the wildtype when the mean±SD values were compared between them. In each casethe difference was statistically significant. It was confirmed, from theresults, that the CNP peptide was overexpressed in the CNP transgenicmouse.

Example 5 Histological Analysis of the Articular Cartilage of CNPTransgenic Mouse

To perform histological analysis of the articular cartilage for thethickness and the number of chondrocytes, 5 each of 9-week-old femaleCNP transgenic mice and female normal litter of mice were euthanized byexsanguination from the postcava under ether anesthesia, and the femurwas fixed in 20% formalin for a week. After dipping in a 20% aqueoussolution of EDTA-4Na (pH 7.4) (Junsei Chemical Co., Ltd., Japan) fordecalcification, the facies patellaris femoris was subjected to amidline sagittal section and embedded in paraffin by a conventionalmethod to prepare paraffin blocks. A 4 mm-thick section was furthersectioned with a microtome to prepare paraffin sections, which werestained with hematoxylin-eosin stain. For the thickness of the articularcartilage, one microscopic field observed using an objective lens (×10)was incorporated into an image analysis software (IPAP, SumikaTechnoservice, Japan), and the length was measured at five points in thefield using the software to calculate an average of the length, whichaverage was used as the thickness of the articular cartilage of theindividual. The same field was measured for the number of chondrocytesas well. Mean values and standard deviations for these items werecalculated in normal mice and CNP transgenic mice of the same sex(Microsoft Excel 2000, Microsoft), and statistical analysis wasperformed using the unpaired Student's t-test (SAS ver.6.12, SASInstitute Japan, Japan).

CNP transgenic mice, both male and female mice, demonstrated that theyhad statistically significantly thicker articular cartilage (FIG. 4). Inaddition, the number of articular chondrocytes per microscopic field wasshown to be statistically significantly larger in both male and femaleCNP transgenic mice (FIG. 5).

These results revealed that GC-B (NPR-B) activating substances such asCNPs can increase the thickness of the articular cartilage by increaseof the number of chondrocytes, as well as by increase of the cell volumedue to hypertrophy of individual chondrocytes as generally known [J BiolChem 2003; 278(21): 18824-32].

Example 6 Resistance of CNP Transgenic Mouse to Osteoarthritis Model

An osteoarthritis animal model was created by injecting collagenase intothe knee joint to destabilize the knee joint ligament and meniscus (Am.J. Pathol. 1989; 135:1001-14). Resistance to arthritis and articularcartilage degeneration was evaluated in this animal model using a CNPtransgenic mouse to confirm the preventive and therapeutic effects ofCNPs on osteoarthritis. 6 μl of 3% type II collagenase (Sigma) solutionin physiological saline was injected twice (initial dosing day and after7 days) into the right knee joint of CNP transgenic mice and the litterof wild-type C57BL/6 strain mice. The width of both the right and leftknee joints was measured on time with a slide caliper (Mitutoyo Corp.,Japan) for 28 days after administration, and the difference between theright and left knee joints was calculated to represent the swelling ofknee joints. The area under the time-course curve (AUC) of sequentialchanges was calculated by the trapezoidal method, and compared by theStudent's t-test between the CNP transgenic mouse and the wild-typemouse. The result that the AUC was significantly smaller in the CNPtransgenic mouse than in the wild type indicates that the CNP transgenicmouse is resistant to knee joint swelling caused by collagenase (FIG.6). To perform histopathological evaluation of the arthritis andarticular cartilage degeneration, the knee joint was removed followingeuthanasia by exsanguination under ether anesthesia on day 28 after theadministration of collagenase, and hematoxylin-eosin-stained andsafranine O-stained samples were prepared as described in Example 5, andanalyzed histologically. As a result, the wild-type mouse showedcollagenase-induced marked synovial cell growth, granulation andinflammatory cellular infiltration in the synovial membrane while thesechanges were remarkably reduced in the CNP transgenic mouse (FIG. 7).For the degeneration of the articular cartilage, the wild-type mouseshowed decreased safranine O stainability and a decreased proteoglycancontent in the articular cartilage while these changes were mild in theCNP transgenic mouse, and this provides histopathological evidence thatthe CNP transgenic mouse is resistant to the degenerative changes inarticular cartilage caused by administration of collagenase (FIG. 8).The plasma CNP level as determined using a EIA kit (PhoenixPharmaceutical) was an average of 0.21 ng/mL in the wild-type mouse and0.50 ng/mL in the CNP transgenic mouse.

These results revealed that CNPs have inhibitory action on arthritis anddegenerative changes in articular cartilage in osteoarthritis.

Example 7 Therapeutic Effect of CNP Infusion on Osteoarthritic Model (1)

An osmotic pump (2004 model, Durect) containing the solutions below wastransplanted subcutaneously in the back of a 9-week-old male C57BL/6 Jstrain mouse.

Solvent: Distilled water containing 5% dextrose (Junsei Chemical Co.,Ltd., Japan), 10% mannose (Nacalai Tesque Inc., Japan) and 5 mmol/Lhydrochloric acid (Wako Pure Chemical Industries, Japan).

10 μg/mL solution (60 ng/day) of CNP-22 (Calbiochem Novabiochem).

100 μg/mL solution (600 ng/day) of CNP-22 (Calbiochem Novabiochem.).

Six days after transplantation, 6 μL of 1.5% type II collagenase (Sigma)solution was injected into the right knee joint, the breadth of both theright and left knee joints was measured on time with a slide caliper(Mitutoyo Corp., Japan) for 28 days after injection, and the differencebetween the right and left knee joints was calculated. This differencerepresented the swelling of knee joints, and the AUC was comparedbetween the solvent control and CNP groups by the Student's t-test (SASver. 6.12). Results showed that the AUC value was significantly lower inthe CNP-22 group at either dose compared to the solvent control group.Hematoxylin-eosin-stained and safranine O-stained samples were preparedaccording to the method as described in Example 5, and analyzedhistopathologically.

As a result, the solvent control group showed collagenase-induced markedsynovial cell growth, granulation and inflammatory cell infiltration inthe synovial membrane while these changes were remarkably reduced in theCNP group (FIG. 9). These results from synovial tissues revealed thatCNPs have a therapeutic effect on osteoarthritis.

Example 8 Therapeutic Effect of CNP Infusion on Osteoarthritic Model (2)

An osmotic pump (2004 model, Durect) containing the solutions below wastransplanted subcutaneously in the back of a 9-week-old male C57BL/6 Jstrain mouse (CLEA Japan, Japan).

Solvent: Distilled water containing 5% dextrose (Junsei Chemical Co.,Ltd., Japan), 10% mannose (Nacalai Tesque Inc., Japan) and 5 mmol/Lhydrochloric acid (Wako Pure Chemical Industries, Japan).

10 mg/mL solution (60 ng/day) of CNP-22 (Calbiochem Novabiochem).

100 mg/mL solution (600 ng/day) of CNP-22 (Calbiochem Novabiochem).

On the following day of transplantation, the mouse was anesthetized withether and subjected to the surgical procedures of anterocrucial ligamentexcision, medial collateral ligament excision and medial meniscus totalresection in the right knee joint to induce osteoarthritis. The breadthof both the right and left knee joints was measured on time with a slidecaliper (Mitutoyo Corp.) for 11 days after administration, and thedifference between the right and left knee joints was calculated. Thisdifference represented the swelling of knee joints, and the AUC wascompared between the solvent control and CNP groups by the Student'st-test (SAS Preclinical Package, SAS Institute Japan, Japan). Resultsshowed that the AUC value was significantly lower in the CNP-22 group ateither dose compared to the solvent control group (FIG. 10).

The results revealed that CNPs are also effective in inhibitingarthritis in osteoarthritis caused by physical overload on the kneejoint resulting from surgical procedures.

Example 9 Combined Effect of Nonsteroidal Anti-Inflammatory Drug (NSAID)and CNP in Collagenase OA Model

An osmotic pump (2004 model, Durect) containing the solutions below wastransplanted subcutaneously in the back of a 9-week-old male C57BL/6 Jstrain mouse.

Solvent: Distilled water containing 5% dextrose (Junsei Chemical Co.,Ltd., Japan), 10% mannose (Nacalai Tesque Inc., Japan) and 5 mmol/Lhydrochloric acid (Wako Pure Chemical Industries, Japan).

1 μg/L solution (6 ng/day) of CNP-22 (Calbiochem Novabiochem).

In addition, to examine the effect of the NSAID indomethacin (Sigma)when used alone and in combination with the CNP, an indomethacinsuspension in 0.2% carboxymethyl cellulose (Nacalai Tesque Inc., Japan)was administered orally in a forced manner at 1 mg/kg once a day for 4successive days from the date of pump transplantation described above.

The experimental groups were set as follows.

Solvent control (infused solvent, orally administered solvent)

CNP 6 ng/day

Indomethacin 1 mg/kg

CNP 6 ng/day+indomethacin 1 mg/kg

On the date of pump transplantation and the following day, 6 μL of 0.15%type II collagenase (Sigma) and 6 μL of 1.5% type II collagenasesolutions, respectively, were injected into the right knee joint, thebreadth of both the right and left knee joints was measured daily with aslide caliper (Mitutoyo Corp., Japan) for 7 days after injection, andthe difference between the right and left knee joints was calculated.This difference represented the swelling of knee joints, and the AUC wascompared between the solvent control and CNP groups by the Student'st-test (SAS ver. 6.12).

As a result, indomethacin when used alone was not inhibitory for theswelling of knee joints. The group given the CNP at 6 ng/daysignificantly inhibited the swelling of knee joints. The group given thecombination of CNP and indomethacin showed significantly strongerinhibition for the swelling of knee joints compared to the group giventhe CNP alone (FIG. 11). These results revealed that the CNP when usedalone is significantly more effective in inhibiting the swelling of kneejoints compared to the NSAID, which is a standard anti-arthritis, andalso has a synergistic effect when used in combination with the NSAID.

Example 10 Effect of CNPs on Adjuvant Arthritis Rat Model

An osmotic pump (2004 model, Durect) containing the solutions below wastransplanted subcutaneously in the back of a 6-week-old male LEW/Crjstrain rat (Charles River Laboratories Japan, Inc., Japan).

Solvent: Distilled water containing 5% dextrose (Junsei Chemical Co.,Ltd., Japan), 10% mannose (Nacalai Tesque Inc., Japan) and 5 mmol/Lhydrochloric acid (Wako Pure Chemical Industries, Japan).

10 μg/mL solution (60 ng/day) of CNP-22 (Calbiochem Novabiochem).

On the following day of transplantation, powder of killed tuberculosisbacteria (M. TUBERCULOSIS DES. H37 RA, DIFCO LABORATORIES) was suspendedin liquid paraffin (Junsei Chemical Co., Ltd., Japan) at a concentrationof 3 mg/mL, and 50 μL was inoculated into the skin at the root of a rattail. After inoculation, the conditions of the limb ends were evaluatedaccording to the following criteria daily using a scoring system, andthe sum of scores for the limb ends was calculated to represent as thearthritis score of the individual.

Score 0: No lesion

Score 1: Flare/swelling is observed in one or more finger joints. Orreddening occurs in the back of the paw with no swelling.

Score 2: Mild swelling occurs in the back of the forelimb or hindlimb.

Score 3: Severe swelling occurs in the back of the forelimb or hindlimb,but not in all fingers.

Score 4: Severe swelling occurs in the back and fingers of the forelimbor hindlimb.

Results showed that the arthritis score was somewhat lower in the CNPgroup than in the solvent control group (FIG. 12A).

Changes in body weight were also measured on a daily basis. Resultsshowed that body weight increased significantly in the CNP groupcompared to the solvent control group (FIG. 12B).

These results revealed that CNPs also inhibit arthritis and improvegeneral condition in an adjuvant rat model.

Example 11 Effect of CNPs on Collagen Arthritis Rat Model

An osmotic pump (2004 model, Durect) containing the solutions below wastransplanted subcutaneously in the back of a 10-week-old female DA/Sicstrain rat (Japan SLC, Inc., Japan).

Solvent: Distilled water containing 5% dextrose (Junsei Chemical Co.,Ltd., Japan), 10% mannose (Nacalai Tesque Inc., Japan) and 5 mmol/Lhydrochloric acid (Wako Pure Chemical Industries, Japan).

1 mg/mL solution (6 μg/day) of CNP-22 (Calbiochem Novabiochem).

Immediately after transplantation, bovine type II collagen (CollagenTechnology Training Co., Japan) was dissolved in 0.1 mol/L aqueousacetic acid so as to make 1.5 mg/mL and suspended in an equal volume ofFreund Incomplete Adjuvant (DIFCO LABORATORIES), and 400 μL of thesuspension was inoculated into the skin on the back of a rat. Changes inbody weight were also measured on a daily basis. In addition, changes inbody weight were also measured in a normal group receiving neither pumptransplantation nor inoculation.

As a result, body weight decreased significantly in the solvent controlgroup compared to the normal group while body weight loss in the CNPgroup was significantly smaller compared to the solvent control group(FIG. 13). These results revealed that CNPs improve general conditionsin a collagen arthritis rat model.

INDUSTRIAL APPLICABILITY

Because the therapeutic or prophylactic agents according to the presentinvention containing a GC-B activator as an active ingredient canincrease the thickness of the articular cartilage and the number ofarticular chondrocytes, provide resistance to articular swelling,inhibit degenerative changes in articular cartilage, provide markedlydecreased changes in synovial cell growth, granulation and inflammatorycellular infiltration, and avoid a decrease in the proteoglycan contentin the articular cartilage, they are useful for the treatment orprevention of arthritis including osteoarthritis, such as degenerativegonarthrosis, degenerative coxarthrosis, elbow osteoarthritis, spinalosteoarthritis and temporomandibular arthrosis. Administration of thepharmaceutical composition according to the present invention can resultin the inhibition of a reduction in, or the regeneration of, thearticular cartilage matrix and chondrocytes in the affected jointportion, and inhibit degenerative changes in articular cartilage andswelling in the articular part, resulting in the inhibition or reductionof arthritic diseases. In particular, because the therapeutic agents forosteoarthritis of the present invention incur less burden and pain onthe patient compared to conventional orthopedic operations, such asarthroscopic surgery, artificial joint substitution and osteotomy, theyprovide superior therapeutic agents with satisfactory QOL for thepatient.

The new finding that GC-B activators have the efficacy as describedabove means that it is possible to inhibit arthritis and promote thegrowth of articular chondrocyte by activating GC-B. In addition, it isalso possible to screen articular chondrocyte growth promoters andtherapeutic agents for arthritis by using the GC-B activity (forexample, the amount of intracellular cGMP produced) as an indication.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

Free Text of Sequence Listing

Description in SEQ ID NO: 1: A disulfide bond is formed between 6-Cysand 22-Cys.

Description in SEQ ID NO: 2: A disulfide bond is formed between 37-Cysand 53-Cys.

Description of artificial sequence in SEQ ID NO: 3: CNP-22 derivative,where a disulfide bond is formed between 6-Cys and 22-Cys.

Description of artificial sequence in SEQ ID NO: 4: CNP-22 derivative,where a disulfide bond is formed between 6-Cys and 22-Cys.

Description of artificial sequence in SEQ ID NO: 5: CNP-22 derivative,where a disulfide bond is formed between 6-Cys and 22-Cys.

Description of artificial sequence in SEQ ID NO: 6: CNP-22 derivative,where a disulfide bond is formed between 1-Cys and 17-Cys.

Description of artificial sequence in SEQ ID NO: 7: CNP-22 derivative,where a disulfide bond is formed between 7-Cys and 23-Cys.

Description of artificial sequence in SEQ ID NO: 8: CNP-22 derivative,where a disulfide bond is formed between 6-Cys and 22-Cys.

Description of artificial sequence in SEQ ID NO: 9: CNP-22 derivative,where a disulfide bond is formed between 1-Cys and 17-Cys.

Description of artificial sequence in SEQ ID NO: 10: CNP-22 derivative,where 4-Xaa=Leu, Ile, Val; 5-Xaa=Lys, Leu, Met; 6-Xaa=Leu, Ile, Ala,Val; 11-Xaa=Ser, Ala, Gly, Thr, Asn; 12-Xaa=Met, Ala, Trp, H is, Lys,Ser, Gly; 14-Xaa=Gly, Lys, Ala, Leu; 15-Xaa=Leu, Met and where adisulfide bond is formed between 1-Cys and 17-Cys.

1. A therapeutic or prophylactic agent for arthritis comprising a guanylcyclase B (GC-B) activator as an active ingredient.
 2. The therapeuticor prophylactic agent according to claim 1, wherein the arthritis isosteoarthritis.
 3. The therapeutic or prophylactic agent of claim 2,wherein the osteoarthritis is osteoarthritis of weight bearing joints ornon-weight bearing joints.
 4. The therapeutic or prophylactic agent ofclaim 3, wherein the osteoarthritis is degenerative gonarthrosis.
 5. Thetherapeutic or prophylactic agent of claim 3, wherein the osteoarthritisis degenerative coxarthrosis.
 6. The therapeutic or prophylactic agentof claim 3, wherein the osteoarthritis is temporomandibular arthrosis.7. The therapeutic or prophylactic agent of claim 1, wherein thearthritis is caused by rheumatoid arthritis.
 8. The therapeutic orprophylactic agent of claim 1, wherein the arthritis is caused byosteoarthritis.
 9. The therapeutic or prophylactic agent of claim 1,wherein the GC-B activator is a C-type natriuretic peptide (CNP) or aderivative thereof.
 10. The therapeutic or prophylactic agent of claim9, wherein the CNP is selected from CNP-22 and CNP-53 from mammals,including human, or birds.
 11. The therapeutic or prophylactic agent ofclaim 9, wherein the CNP is CNP-22 of SEQ ID NO:1 or CNP-53 of SEQ IDNO:2.
 12. The therapeutic or prophylactic agent of claim 9, wherein thederivative has a deletion, substitution or addition of one or severalamino acids in the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2,while possessing a CNP activity.
 13. The therapeutic or prophylacticagent of claim 1, further comprising at least one nonsteroidalanti-inflammatory drug.
 14. An agent for promoting the growth ofarticular chondrocyte, comprising a GC-B activator as an activeingredient.
 15. The agent of claim 14, wherein the GC-B activator is aCNP or a derivative thereof.
 16. The agent of claim 15, wherein the CNPis CNP-22 or CNP-53 from mammals, including human, or birds.
 17. Theagent of claim 15, wherein the CNP is CNP-22 of SEQ ID NO:1 or CNP-53 ofSEQ ID NO:2.
 18. The agent of claim 15, wherein the derivative has adeletion, substitution or addition of one or several amino acids in theamino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, while possessing aCNP activity.
 19. The agent of claim 14, further comprising at least onenonsteroidal anti-inflammatory drug.
 20. A method for inhibitingarthritis, wherein the arthritis is inhibited by activating GC-B. 21.The method of claim 20, wherein the GC-B is activated by a CNP or aderivative thereof.
 22. The method of claim 21, wherein the CNP isCNP-22 or CNP-53 from mammals, including human, or birds.
 23. The methodof claim 21, wherein the CNP is CNP-22 of SEQ ID NO:1 or CNP-53 of SEQID NO:2.
 24. The method of claim 21, wherein the derivative has adeletion, substitution or addition of one or several amino acids in theamino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, while possessing aCNP activity.
 25. The method of claim 20, wherein the GC-B is activatedby a combination of a CNP or a derivative thereof and at least onenonsteroidal anti-inflammatory drug.
 26. A method for promoting thegrowth of articular chondrocyte, wherein said growth is promoted byactivating GC-B.
 27. The method of claim 26, wherein the GC-B isactivated by a CNP or a derivative thereof.
 28. The method of claim 27,wherein the CNP is CNP-22 or CNP-53 from mammals, including human, orbirds.
 29. The method of claim 27, wherein the CNP is CNP-22 of SEQ IDNO:1 or CNP-53 of SEQ ID NO:2.
 30. The method of claim 27, wherein thederivative has a deletion, substitution or addition of one or severalamino acids in the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2,while possessing a CNP activity.
 31. The method of claim 26, wherein theGC-B is activated by a combination of a CNP or a derivative thereof andat least one nonsteroidal anti-inflammatory drug.
 32. A method forscreening an articular chondrocyte growth promoter, comprising screeningcandidate agents for the ability to promote the growth of articularchondrocyte using GC-B activity as an indication.
 33. The method ofclaim 32, comprising preparing cultured cells that express GC-B or cellsfrom articular chondrocytes, culturing the cells in the presence of acandidate agent, and screening the candidate agents for the ability topromote the growth of articular chondrocyte using the cellular GC-Bactivity as an indication.
 34. The method of claim 32 or 33, wherein theGC-B activity is determined as an amount of produced intracellular cGMP.35. The method of claim 32, comprising preparing a cultured cell linewhich has been forced to express GC-B, culturing the cell line in thepresence or absence of a candidate agent, determining the amount ofproduced intracellular cGMP, and screening the candidate agents for theability to promote the growth of articular chondrocyte using as anindication the difference between the amounts of intracellular cGMPproduced in the presence and absence of the candidate agent.
 36. Amethod for screening a therapeutic agent for osteoarthritis, rheumatoidarthritis or other arthritis comprising screening candidate agents foran agent capable of treating osteoarthritis, rheumatoid arthritis orother arthritis using GC-B activity as an indication.
 37. The method forscreening of claim 36, comprising preparing cultured cells that expressGC-B, or cells from articular chondrocytes, culturing the cells in thepresence of a candidate agent, and screening the candidate agent for anagent capable of treating osteoarthritis, rheumatoid arthritis or otherarthritis using the cellular GC-B activity as an indication.
 38. Themethod of claim 36, wherein the GC-B activity is determined as an amountof intracellular cGMP produced.
 39. The method of claim 36, comprisingpreparing a cultured cell line which has been forced to express GC-B,culturing the cell line in the presence or absence of a candidate agent,determining the amount of intracellular cGMP produced, and screening thecandidate agents for an agent capable of treating osteoarthritis,rheumatoid arthritis or other arthritis using as an indication thedifference between the amounts of intracellular cGMP produced in thepresence and absence of the candidate agent.
 40. A therapeutic orprophylactic agent for osteoarthritis comprising a guanyl cyclase B(GC-B) activator as an active ingredient.
 41. The therapeutic orprophylactic agent for osteoarthritis of claim 40, further comprising atleast one nonsteroidal anti-inflammatory drug.
 42. A therapeutic orprophylactic agent for rheumatoid arthritis comprising a guanyl cyclaseB (GC-B) activator as an active ingredient.
 43. The therapeutic orprophylactic agent for rheumatoid arthritis of claim 42, furthercomprising at least one nonsteroidal anti-inflammatory drug.
 44. Anactivation promoter for a GC-B activator, comprising a nonsteroidalactivator.
 45. The activation promoter of claim 44, wherein the GC-Bactivator is a CNP or a derivative thereof.
 46. The activation promoterof claim 45, wherein the CNP is selected from CNP-22 and CNP-53 derivedfrom mammals, including human, or birds.
 47. The activation promoter ofclaim 45, wherein the CNP is CNP-22 of SEQ ID NO:1 or CNP-53 of SEQ IDNO:2.
 48. The activation promoter of claim 45, wherein the derivativehas a deletion, substitution or addition of one or several amino acidsin the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, whilepossessing CNP activity.
 49. The activation promoter of claim 44,wherein the nonsteroidal activator is a cyclooxygenase inhibitor. 50.The activation promoter of claim 49, wherein the cyclooxygenaseinhibitor is selected from the group consisting of indomethacin,ibuprofen, piroxicam, salicylic acid, diclofenac, ketoprofen, naproxenand piroxicam.
 51. A method for activating a GC-B activator, wherein theactivation promoter of claim 44 is used.